The present invention relates to a method for reversing age-related changes in the lipid composition of heart muscle tissue and other age-related physiological characteristics.
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One of the biochemical changes which occur with aging is a change in membrane lipid composition. In mammalian plasma membranes, the main variation occurs in the relative composition of phosphatidylcholine (PC). which decreases with age, and sphingomyelin (SM) and cholesterol, which increase with age (Barenholz). The changes in the relative amounts of PC and SM is especially great in tissues which have a low phospholipid turnover. For example, plasma membranes associated with the aorta and arterial wall show a 6-fold decrease in PC/SM ratio with aging. SM also increases in several diseases, including atherosclerosis. The SM content can be as high as 70-80% of the total phospholipids in advanced aortic lesion (Barenholz 1982, 1984).
The most striking differences between PC and SM derived from biological membranes are (a) the phase transition temperature of the lipids, and (b) the hydrogen-bonding character of the two lipids in a lipid-bilayer. Most sphingomyelins have transition temperatures in the physiological temperature range between 30xc2x0 and 40xc2x0 C., whereas most naturally occurring phosphatidylcholines are well above their transition temperature at 37xc2x0 C. (Barenholz 1980, 1982, 1984). In terms of hydrogen bonding, the difference in the polar regions of these two lipids enables SM to be both a donor and acceptor of hydrogen in hydrogen bonding, while PC can only serve as a hydrogen donor.
Whether or not related to these differences, the relative content of PC to SM in mammalian plasma membranes appears to affect cell functioning significantly. The inventors and colleagues have recently reported on changes in the lipid composition and activity of primary rat myocytes in culture over time. Measurements of PC and SM content in the cells showed a decline of PC/SM ratio from 5 to about 2 in the first three days in culture, and from 2 to about 1 over the next 14 days in culture. The lipid composition changes were accompanied by a dramatic change in heart cell activity, as measured by the beating rate of the cultured cells. Between days 7 and 12 in culture, the beats/minute fell from about 160 to about 20, and significant increases in the activities of at least seven enzymes, expressed as Vmax/DNA, were also observed. One of these enzymes was creatine phosphokinase (CPK), which plays a major role in intracellular energy transport from mitochondria to myofibrils, and in the regulation of energy production coupled to energy utilization (Yechiel 1985a, 1985b).
The ratio of cholesterol to phospholipid also appears to be an important determinant in regulating the properties of biological membranes (Cooper 1977). Several studies have shown that certain properties of biological membranes can be altered by enrichment with or depletion of cholesterol (Borochov, Cooper 1978, Hasin). In general, there is a strong positive correlation between changes in SM and cholesterol levels in mammalian plasma membranes. That is, changes in the content of one are followed by changes in the other (Wallach, Barenholz 1982, 1984).
It is not clear how cells maintain the various lipid compositions in their different membranes, or why lipid composition changes with aging. In theory, lipid compositional changes could result from changes in the rates of synthesis or degradation of specific lipid components, or changes in the rate of lipid exchange or transfer between serum and the cell membrane. The latter mechanism has received considerable attention with regard to in vitro studies on lipid exchange in cultured biological cells. A number of studies have shown lipid exchange between biological membranes and artificial lipid bilayer vesicles or liposomes (Pagano, Martin, Cooper 1977, Frank). In general, phospholipid exchange between cells and liposomes is accelerated by the presence of a variety of phospholipid transfer proteins, including high and low density serum apolipoproteins (Wirtz, Kader). Cholesterol exchange between biological membranes and liposomes, and/or serum lipoprotein particles is also well known (Hasin, Grunze, Pal).
The ability to alter the lipid composition of biological cells by lipid exchange provides a means for studying the effect of lipid variation on cell function. For example, in the above-discussed myocyte culture system in which a decline in PC/SM ratio over time is accompanied by a drop in beating frequency, it can be asked whether (a) the original lipid composition of the cells can be restored by lipid exchange; and (b) if so, if original cell functioning, i.e., initial beating rate, is also restored. The inventors and coworkers have investigated this question, using small unilamellar PC liposomes as a vehicle for lipid exchange with the cells (Yechiel 1985a, 1985b). The study showed that lipid exchange increased both PC/SM and PC/cholesterol ratios, and thus reversed the normal lipid compositional changes which occur in the cultured cells over time. Interestingly, lipid exchange also restored cell beating frequency to its original levels, with the beating frequency showing a jump from about 20 to 160 within one day of cell exposure to the PC liposomes. Lipid exchange also led to a reduction in cellular enzymes, such as CPK, which were normally increase with time in culture.
According to an important aspect of the present invention, it has been discovered that PC-rich liposomes are able to reverse age-related changes in the lipid composition of heart muscle cells in animals which have received the liposomes by parenteral administration. One significant benefit of the liposome treatment is that the ability to withstand respiratory stress, which normally shows a gradual loss with increasing age (starting above the age of about 15 months in rats), is significantly improved. Furthermore, lipid exchange and concomitant improvement in respiratory hardiness are produced within several days of initial liposome administration. The method is applicable to both veterinary animals and humans.
According to another aspect of the invention, the effect of liposome treatment on heart muscle lipid exchange is reflected in a number of physiological changes which can be readily observed in serum samples from the treated individual (veterinary animal or human). One of these changes is a decrease in the serum creatine phosphokinase (CPK), to levels which are characteristic of relatively younger animals. Typically, serum CPK in a relatively aged animal will fall by 50% or more several days after liposome administration is first begun. Another change which is readily observable in the treated animal is greater tolerance of the red blood cells to osmotic shock. Again this change is seen most strongly in the older animals, with the cells from the treated individual showing an osmotic fragility which is characteristic of the animal at a younger age. The change in osmotic fragility presumably is due to a greater PC/SM and/or phospholipid/cholesterol ratio which occurs in red blood cells as a result of the liposome treatment.
In practicing the method, there is provided a suspension of liposomes containing substantially more PC and substantially less SM than that characteristically found in heart tissue from individuals of about the same age. In one preferred embodiment, the liposomes are small unilamellar vesicies (SUVs) having sizes predominantly between 0.02 and 0.08 microns, and composed predominantly or exclusively of purified PC such as egg PC. The liposome suspension is administered in an amount which is effective to produce, over a period of at least several days, a substantial decrease in the level of serum CPK in the treated animal. The course of treatment can be followed by monitoring blood CPK or changes in blood cell lipid composition or osmotic fragility.
Another important use of the liposome treatment-method is for increasing longevity in the treated individual. Studies on laboratory animals indicate that treating relatively aged animals with liposomes over an extended period increases animal lifespan by an average of about 36%.
Still another important use of the method is for increasing male sexual competence. Treating relatively old lab animals with liposomes according to the invention reversed the near-complete loss of competence normally seen in the older male animals. The method is particularly useful for treating older breeding animals.
Accordingly, it is a general object of the invention to provide a method of treating an aged individual which significantly enhances the animal""s ability to withstand respiratory stress.
A related object of the invention is to provide such a method which reverses age-related lipid composition changes in heart muscle cells.
Another object of the invention is to provide such a method in which the course of treatment can be easily monitored by changes in serum enzyme levels or red blood cell properties.
It is yet another object of the invention is to provide a method which leads to qualitative benefits in aged individuals, including greater longevity and sexual function.
These and other objects and features of the invention will be more fully appreciated from the following detailed description of the invention.
A. Unsized Liposomes
The invention involves, in one aspect, administering liposomes parenterally to an individual (a veterinary animal or human) to reverse age-related changes in the lipid composition of organs and tissues. such as heart muscle cells and red blood cells, by lipid exchange. Since the aging process in heart muscle is characterized in decrease in PC, and a concomitant increase in SM and cholesterol, the liposomes are designed to promote exchange of PC from liposomes to heart cell membrane, and exchange of SM from the heart muscle to the liposomes. The liposomes are also preferably designed to promote cholesterol exchange from the organs and tissues, such as heart muscle cells and red blood cells, to the liposomes.
In order to promote the desired lipid exchange. the mole percent of liposomal PC is substantially greater than, and the mole percent of SM is substantially less than, that of heart tissue from the treated individual, i.e., the PC and SM levels characteristic of individuals of the same age, species, and sex. Preferably the liposomes contain at least about 25 mole percent more PC and at least about 10 mole percent less SM than the heart muscle cells in the treated individual. In one preferred liposome preparation described and used in Examples I-VIII, the liposomes are formed of substantially pure PC. These liposomes act to increase the PC/SM ratio of the heart cells, and to lower cholesterol levels, as will be seen in Example II. The extend of cholesterol reduction can be modulated, from maximum reduction to virtually no reduction, by increasing the amount of cholesterol from zero up to a mole percentage comparable to that in the heart muscle cells of the treated individual.
Another important consideration in the selection of liposome lipids is the acyl chain composition of the phospholipids. As indicated above, the shift from PC to SM phospholipid which occurs with age is also accompanied by an overall increase in the saturation and chain length of the acyl chain moieties of the membrane phospholipids. It is therefore preferred that the PC component of the lipids have an acyl chain composition which is characteristic, at least with respect to transition temperature, of the acyl chain components in heart cells from the animal at a younger age. One preferred PC composition is egg PC, which contains predominantly 1-palmitoyl,2-oleyl PC and 1-palmitoyl,2-linoleyl PC.
The liposomes may contain other lipid components, as long as these are not immunogenic and do not inhibit the desired lipid exchange between the liposomes and heart muscle cells. Additional components may include negatively charged lipids, such as phosphatidylglycerol (PG) or phosphatidylserine (PS). Of course, the mole percentage of these lipids should be relatively low with respect to PC. Lipid protective agents, such as (xcex1-tocopherol (xcex1-T), xcex1-tocopherol acetate, or xcex1-tocopherol succinate, may also be included in the lipids forming the liposomes, to protect the lipid components against free radical damage (Levida). Typically such agents are included at a mole percentage between about 0.5% and 2%. It is advantageous to add xcex1-T to the liposomes to maintain a balance between vitamin E and polyunsaturated lipids in the liposomes.
A variety of methods for producing liposomes are available, and these have been extensively reviewed (Szoka 1980). In general these methods produce liposomes with heterogeneous sizes from about 0.02 to 10 microns or greater. Since, as will be discussed below, liposomes which are relatively small and well defined in size are preferred for use in the present invention, a second processing step for reducing the size and size heterogeneity of liposomal suspensions will usually be required.
In one preferred method for forming the initial liposome suspension, the vesicle-forming lipids are taken up in a suitable organic solvent system, and dried in vacuo or under an inert gas to form a lipid film in a vessel. An aqueous suspensions medium, such as a sterile saline solution, is added to the film, and the vessel is agitated until the lipids have hydrated to completion, typically within 1-2 hours. The amount of aqueous medium added is such as to produce a final liposome suspension containing preferably between about 10 and 30 g lipid per 100 ml.
The lipids hydrate to form multilamellar vesicles (MLVS) whose sizes range between about 0.5 microns to about 10 microns or larger. In general, the size distribution of MLVs can be shifted toward slightly smaller sizes by hydrating the lipids under more vigorous shaking conditions. Example I describes the preparation of egg PC MLVs, prior to treating the MLVs with ultrasonic irradiation to reduce the liposome sizes.
The aqueous medium used in forming the liposomes may contain water-soluble agent(s) which enhance the stability of the liposomes on storage. A preferred stabilizing agent is an iron-specific trihydroxamine chelating agent, such as desferrioxamine. The combination of a lipophilic free-radical quencher, such as xcex1-T, and the water-soluble chelator gave substantially better protection against lipid peroxidation damage than did either protective agents alone. The chelator is included in the aqueous medium in molar excess of the amount of free iron in the medium. Typically, a chelator concentration of between about 10-50 xcexcM is sufficient.
B. Sizing Liposomes
The liposome suspension may be sized to achieve a selective size distribution of vesicles in a size range less than about 1 micron and preferably less than about 0.2-0.3 microns. Liposomes in this size range can be readily sterilized by filtration through a depth filter. Smaller vesicles also show less tendency to aggregate on storage, thus reducing potentially serious vascular blockage problems when the composition is administered parenterally. Finally, liposomes which have been sized down to the submicron range show more uniform biodistribution and drug clearance characteristics.
Several techniques are available for reducing the sizes and size heterogeneity of liposomes, in a manner suitable for the present invention. Ultrasonic irradiation of a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles (SUVs) between about 0.02 and 0.08 microns in size. A sonicating procedure used to produce SUVs is described in Example I. Homogenization is another method which relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, MLVs are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination.
Extrusion of liposomes through a small-pore polycarbonate membrane is an effective method of reducing liposome sizes down to a relatively well defined size distribution whose average in the range between about 0.03 and 1 micron, depending on the pore size of the membrane. Typically, the suspension is cycled through the membrane several times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller pore membranes, to achieve a gradual reduction in liposome size.
More recently, it has been discovered that a suspension of liposomes having heterogeneous sizes above and below 1 micron can be sized efficiently by passage through an asymmetric ceramic filter. A preferred ceramic filter is a Ceraflow Microfilter, 0.2-1 xcexc inner-surface pore size, available commercially from the Norton Company (Worcester, Mass.), and supplied as a multifilter cartridge-type filter apparatus. This sizing method is described in U.S. patent application for xe2x80x9cLiposome Extrusion Methodxe2x80x9d, filed Feb. 13, 1986.
Centrifugation and molecular sieve chromatography are other methods which are available for producing a liposome suspension with particle sizes below a selected threshold less than 1 micron: These two methods both involve preferential removal of larger liposomes, rather than conversion of large particles to smaller ones. Liposome yields are correspondingly reduced.
The size-processed liposome suspension may be readily sterilized by passage through a sterilizing membrane having a particle discrimination size of about 0.2 microns, such as a conventional 0.22 micron depth membrane filter. If desired, the liposome suspension can be lyophilized for storage and reconstituted shortly before use.
A. Liposome Administration
In one treatment method, the liposomal suspension is administered parenterally in one or more doses until a desired change in the lipid composition of heart muscle cell is produced. Changes in lipid composition in heart muscle cells are accompanied by changes in serum CPK and in red blood cell properties, allowing the course of treatment to be monitored readily by blood sampling.
The liposomes may be conveniently administered as a series of dosages, given over a period of at least several days, and preferably maintained by continued doses at one to several month intervals over the lifetime of the treated individual. The amount of liposomes administered at each dose is preferably between about 0.01 and 1.0 g per kg of body weight and may be substantially less. A typical dose for an 80 kg individual would be between about 40 and 80 grams lipid, corresponding to between 200 and 400 ml of an up to 20% liposome suspension. Administration may be by iv injection, but is preferably done by iv drip over a period of at least about 1 hour, to minimize tissue and blood trauma at the site of administration. The liposomes may be suspended in sterile saline or in a nutritional or drug-containing medium, such as a glucose/salt medium, to combine liposome treatment with other parenteral therapy.
Before the first dose is given, a blood sample is taken for determination of serum enzyme and/or blood cell characteristics which will be monitored during the course of the liposome treatment. As will be discussed below, liposome treatment produces a readily measurable drop in serum CPK level, to a level characteristic of the individual at a much younger age. The fall in CPK presumably reflects a reversal in age-related lipid composition in muscle cells, as documented in Examples II and III, respectively. The liposome treatment is continued until a drop of at least about 25% in serum CPK, and preferably 50% or more, is observed. In the study described in Example II, serum CPK levels declined to about 15% of their pre-treatment values after nine days of treatment.
Liposome treatment also produces easily observable changes in the lipid composition and osmotic fragility of red blood cells. These changes are documented in Examples IV and V below. Osmotic fragility can be determined using simple spectrophotometric techniques to measure percent hemoglobin release when the packed cells are suspended in one of a series narrowly graded salt solutions. The method is described in Example V.
The change in lipid composition of red blood cells can be determined by first extracting blood-cell lipids, then separating selected lipid components by conventional chromatographic methods. PC and SM can be separated readily by thin layer chromatography (TLC) as described in Examples I and IV, for determining the PC/SM ratio. An advantage of following blood cell PC/SM ratio changes to monitor the course of liposome treatment is that relatively large PC/SM changes are observed. For example, in the 9-day treatment described in Example IV, the PC/SM ratio in red blood cells in 18-month old rats increased nearly 7-fold.
Where the therapeutic liposomes are also designed to promote cholesterol exchange from tissues and organs, such as heart tissue cells, to the liposomes (by virtue of low cholesterol content in the liposomes). the course of therapy may also be monitored by following the changes in the cholesterol content. Methods for measuring and expressing changes in membrane cholesterol are detailed in Examples I and III. Changes in phospholipid composition in erythrocytes during liposome treatment are generally more modest than those in PC/SM.
Following the first liposome administration, the levels of serum CPK (or red blood cell characteristic) are measured, and a second liposome dose is given typically 2 and 7 days after the first dose. Further doses may likewise be given at 2-7 day intervals until the serum property being measured begins to plateau. Thereafter, the individual can be maintained at the desired lipid composition state by periodic maintenance. liposome treatments, e.g., every 1-2 months. Example VIII illustrates a treatment regimen in which lab animals were treated initially with two liposome doses spaced a week apart, then maintained with a single liposome injection every two months. The animals showed at least a 36% increase in longevity over untreated animals.
B. Biochemical Effects
The liposome treatment described above was tested in laboratory animals, to determine its effectiveness in reversing age-related changes in the lipid composition of heart muscle cells, and increasing resistance to respiratory stress. In one series of tests, 18 month old rats were treated with three liposome doses, administered every three days for six days (three injections), and the animals were sacrificed three days after the final injection. The PC/SM and cholesterol content of heart muscle cells was determined as detailed in Example II. The values were compared with those obtained from relatively young animals (three months old) and from untreated 18 month old animals. The results show that both the more than twofold decrease in PC/SM ratio and the more than threefold increase in cholesterol content which occur normally between ages three and eighteen months were completely reversed by the liposome treatment.
The three groups of animals were also tested for heart muscle and serum CPK levels, as described in Example III. The changes in lipid composition which normally occur between three and eighteen months in rats is accompanied by approximately threefold increases in both heart muscle cell and serum CPK. After nine days of liposome treatment, heart cell CPK declined about threefold to levels normally seen in 3 month old animals, and serum CPK declined eightfold to a level substantially lower than that in 3 month old animals. The dramatic fall in serum CPK in treated animals thus provides a sensitive indicator of heart lipid changes occurring during liposome treatment. The concomitant reduction in heart muscle CPK during treatment indicates that the fall in serum CPK is in part due to declining levels of heart muscle CPK.
The above-noted changes in heart cell lipid composition were measured on whole heart homogenates, and therefore represent lipid contributions from both myocardial (heart) cells and connective fibroblasts. To confirm that the observed change in lipid composition also reflects changes in myocardial cells, heart cells from three month old and eighteen month old animals were isolated, cultured under conditions which lead to myocardial reaggregates, then tested for lipid exchange with egg PC liposomes. The methods and results are detailed in Example VI. The reaggregates showed a decrease in PC/SM ratio and an increase in cholesterol level, when comparing cells from three and eighteen month old animals, and these age-related changes were substantially reversed by incubation with the egg PC liposomes. The results indicate that the observed lipid effects seen in heart tissue in vivo are due at least in part to changes in myocardial membrane lipids.
Red blood cells from each of the three groups of animals were tested for changes in red blood cell lipid composition and osmotic fragility. The results are discussed above and in Examples IV and V. Briefly, liposome more than reversed age-related changes in PC/SM ratio and cholesterol levels which normally occur between three and eighteen months of age, both increasing the PC/SM ratio and decreasing the cholesterol level with respect to erythrocytes from three month old animals. The change produced in lipid composition is reflected by ability to withstand greater osmotic shock.
A. Increased Resistance to Respiratory Stress
An important therapeutic application of the present invention is increasing an individual""s ability to withstand cardiac stress. This application is valuable for individuals who have suffered cardiac trauma, such a myocardial infarction, or who are at high risk of heart trauma. In either case, additional stress on the heart, in the form of increased oxygen demand or elevated blood pressure can cause cardiac failure or serious damage to the heart.
The utility of the treatment has been shown in laboratory animals. Here the animal""s ability to withstand cardiac stress before and after treatment was measured by a standard lab procedure, in which an animal is place in a defined-volume chamber which does not allow gas exchange with the outside. During the course of the test, the depletion of oxygen and accumulation of carbon dioxide reduces the animal""s blood pressure gradually to near-zero levels. The ability to withstand respiratory stress is measured by the time in the chamber before the animal""s blood pressure drops to near zero. The test results are reported in example VII. After three liposome treatments, and nine days after the first treatment, 18 month old male rats were able to maintain blood pressure about 50% longer than untreated rats. The treated rats also showed a much slower rate of increase of serum CPK during the test than untreated animals. Blood monitoring throughout the test period showed that both treated and untreated animals maintained comparable levels of blood oxygen and carbon dioxide, indicating that the better performance of treated animals was not merely a blood-gas content effect.
The inventors and coworkers have previously investigated the relationship between membrane lipid composition and a number of biological properties of rat myocytes in culture,including changes in the level of a number of enzymes, cell morphology, and the beating rate of heart myocytes. The studies, which are mentioned above, showed that the PC/SM ratio of the cells declined severalfold over a fourteen-day culture period and cholesterol content/DNA increased about 1.6-fold in the same period. The addition of PC SUVs to the culture reversed all of the age-related changes in the cell, including membrane lipid composition, enzyme levels, cell morphology, and heart activity, as measured by beat frequency. In particular, the rapid restoration of initial cell beating rate with addition of liposome suggest that lipid composition is a critical factor in heart muscle cell performance, it is likely, therefore, that the increased ability to withstand respiratory shock seen in liposome-treated animals is due, at least in part, to enhanced heart performance.
The treatment procedure generally follows the liposome administration regimen outlined above, in which the individual is given an initial dose of preferably between about 0.5-2 g lipid/kg body weight, and one or more subsequent dose every 2-7 days thereafter, at a long-term dose rate of between about 0.001-1 g lipid/kg animal weight per day. A maintenance dose of between about 0.1-1 g lipid/kg administered every 1-2 months may be employed. The liposome used in the treatment are formulated to promote lipid exchange of PC from the liposomes into the heart cells, and SM exchange in the opposite direction, as described above. The liposomes are also preferably formulated to promote cholesterol exchange from the cells to liposomes, and one preferred liposome formulation is composed of pure egg PC. However, it may be advisable in long-term treatment to include cholesterol in the liposomes, to prevent too much cholesterol depletion in the red blood cells.
Biochemical changes in lipid composition of the heart are monitored, as above, by measuring related changes in serum CPK and/or changes in red blood cell lipid composition or osmotic fragility. Heart functioning can be monitored during the treatment period by conventional EKG.
B. Increased Animal Longevity
According to another aspect of the invention, it has been discovered that laboratory animals which have received liposome treatment, as described above, live significantly longer than untreated animals. The results of a study on longevity of male laboratory rats is described in Example VII. Briefly, 30 month old rats were given an initial injection of liposomes, followed by a second injection 1 week later, and maintenance injections every two months. A group of untreated rats died between ages 32 and 38 months, with an average age of death of about 34 months. The group of treated animals were sacrificed between ages 42 and 48 months.
It is interesting to note that longevity was extended in the treated animals, even though treatment was not begun until a relatively advanced age, i.e., within a few months of the time the animals would normally have died. This finding indicates that liposomes are effective in reversing age-related changes in lipid composition, even at an advanced age. The approximately 36% increase in longevity indicates that the alteration in lipid composition produced by liposome-treatment confers widespread physiological benefits (including, presumably, increased cardiac performance) which are related to longevity. The increase in longevity which is achievable can be appreciated from the projected result in humans. Assuming that a human lives on the average about 2 years for each month of a laboratory rat, liposome treatment would result in a major increase of longevity.
C. Increased Male Sexual Competence
Another benefit conferred by liposome treatment is an apparent increase in fertility of treated males. In the study reported in Example VIII, male lab rats 30 months and older received three liposome doses over a 6 day period. Normally male rats at this age are unable to sire litters when placed in the same cage with younger, otherwise fertile female rats. For example, when ten untreated rats of this age were each housed with three female rats, each 5-6 months old, only two out of the thirty females had litters, and in each case, the litter was smaller than the usual 10-13 animal litter sired by younger males. Treated rats, by contrast, showed normal male fertility. All of the rats sired litters in all three females, and all of the litter sizes were the normal 10-14 size.
The treatment regimen in animals preferably involves a series of liposome injections several days to weeks before mating activity. The course of the treatment can be followed, as above, by monitoring the change in serum CPK or change in erythrocyte properties, as discussed. The treatment is expected to be especially useful in horse and cattle breeding, where extending the breeding life of selected animals would be valuable.
The following examples illustrate various aspects and uses of the present invention, but are in no way intended to limit the scope thereof.
Egg phosphatidylcholine (egg PC) and bovine brain sphingomyelin (SM) were prepared according to known methods (Shinitsky, Barenholz 1976). Both lipids were more than 99% pure, based on thin layer chromatography analysis. The egg PC fatty acid composition was similar to the reported composition (Hertz). The main PCs of the preparation included 1-palmitoyl,2-oleyl PC and 1-palmitoyl,2-linoleyl PC. Cholesterol, about 99% pure, was obtained from Sigma (St. Louis, Mo.). Thin-layer chromatography platesxe2x80x940.25 silica gel HR and 0.024 silica gelxe2x80x94were obtained from Merck (Darmstadt, Germany) and Analtech (Newark, Del.), respectively.